Abstract:Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway that is distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which depend on glycolysis. This suggests that increased cellular glucose may prime necroptosis. Here we show that exposure to hyperglycemic levels of glucose enhances necroptosis in primary red blood cells (RBCs), Jurkat T cells, and U937 monocytes. Pharmacologic or… Show more
“…In the present study we have demonstrated a novel shift from caspase-dependent extrinsic apoptosis to RIP1-dependent necroptosis in high glucose conditions. This is distinct from our previous work in which we showed hyperglycemic enhancement of cell death in response to specific stimuli of necroptosis 23 . In those experiments necroptosis was induced in normal and hyperglycemic conditions via artificial inhibition of apoptosis and was shown to increase in hyperglycemic conditions 23 .…”
Section: Discussioncontrasting
confidence: 99%
“…Our previous work established exacerbation of cerebral HI injury in hyperglycemic neonatal mice in a RIP1-dependent manner 23 . Based on our current work, we hypothesized that this may be the result of a shift from apoptosis to necroptosis in vivo .…”
Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR–Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs in vivo. This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.
“…In the present study we have demonstrated a novel shift from caspase-dependent extrinsic apoptosis to RIP1-dependent necroptosis in high glucose conditions. This is distinct from our previous work in which we showed hyperglycemic enhancement of cell death in response to specific stimuli of necroptosis 23 . In those experiments necroptosis was induced in normal and hyperglycemic conditions via artificial inhibition of apoptosis and was shown to increase in hyperglycemic conditions 23 .…”
Section: Discussioncontrasting
confidence: 99%
“…Our previous work established exacerbation of cerebral HI injury in hyperglycemic neonatal mice in a RIP1-dependent manner 23 . Based on our current work, we hypothesized that this may be the result of a shift from apoptosis to necroptosis in vivo .…”
Apoptosis and necroptosis are the primary modes of eukaryotic cell death, with apoptosis being non-inflammatory while necroptosis is highly inflammatory. We previously demonstrated that, once activated, necroptosis is enhanced by hyperglycemia in several cell types. Here, we determine if hyperglycemia affects apoptosis similarly. We show that hyperglycemia does not enhance extrinsic apoptosis but potentiates a shift to RIP1-dependent necroptosis. This is due to increased levels and activity of RIP1, RIP3, and MLKL, as well as decreased levels and activity of executioner caspases under hyperglycemic conditions following stimulation of apoptosis. Cell death under hyperglycemic conditions was classified as necroptosis via measurement of markers and involvement of RIP1, RIP3, and MLKL. The shift to necroptosis was driven by RIP1, as mutation of this gene using CRISPR–Cas9 caused cell death to revert to apoptosis under hyperglycemic conditions. The shift of apoptosis to necroptosis depended on glycolysis and production of mitochondrial ROS. Importantly, the shift in PCD was observed in primary human T cells. Levels of RIP1 and MLKL increased, while executioner caspases and PARP1 cleavage decreased, in cerebral tissue from hyperglycemic neonatal mice that underwent hypoxia-ischemia (HI) brain injury, suggesting that this cell death shift occurs in vivo. This is significant as it demonstrates a shift from non-inflammatory to inflammatory cell death which may explain the exacerbation of neonatal HI-brain injury during hyperglycemia. These results are distinct from our previous findings where hyperglycemia enhanced necroptosis under conditions where apoptosis was inhibited artificially. Here we demonstrate a shift from apoptosis to necroptosis under hyperglycemic conditions while both pathways are fully active. Therefore, while our previous work documented that intensity of necroptosis is responsive to glucose, this work sheds light on the molecular balance between apoptosis and necroptosis and identifies hyperglycemia as a condition that pushes cells to undergo necroptosis despite the initial activation of apoptosis.
“…It is well known that glycolysis regulates the production of ROS, and that ROS and AGEs are effector molecules of necroptosis. 11 WB results showed that the expression of RIP1, RIP3, and p-MLKL protein was increased at 22 mM; however, the expression of ATF4 was decreased ( Figure 3F and 3G). Put together, the results show that the high glucose (22 mM) affected the cell growth of osteoblast cells and induced cell necroptosis.…”
Section: High Glucose Promoted Necroptosis In Osteoblast Cellsmentioning
Diabetes and periodontal diseases have a mutual promoting relationship that induces severe tissue damage and cell death. The potential roles of microRNAs (miRNAs) and the type of cell death involved in diabetes‐associated periodontitis are obscure. The gingival tissues of patients were obtained and MC3T3‐E1 cells were costimulated with high glucose and lipopolysaccharide (LPS). Osseous morphometric analysis was evaluated with micro‐CT, and histological characteristics were measured by hematoxylin/eosin and immunohistochemical staining. Cytokine secretion was confirmed by enzyme‐linked immunosorbent assay, and reactive oxygen species (ROS) was measured using a DCFH‐DA probe kit. Gene expression was measured by real‐time quantitative reverse transcription PCR (qRT‐PCR), and protein expression was assessed by Western blot and immunofluorescence analysis. The miR‐214 level, receptor‐interacting serine‐threonine protein (RIP) 1, RIP3, and phospho‐mixed lineage kinase domain‐like (p‐MLKL) protein expression were elevated in the inflamed gingival tissues of diabetes‐associated periodontitis patients, with activating transcription factor 4 (ATF4) expression showing the opposite effect. The high glucose (22 mM) could not induce significant increase of RIP1, RIP3, and p‐MLKL; however, the high glucose and LPS (500‐1000 ng/mL) cotreatment resulted in increase in the number of RIP1, RIP3, and p‐MLKL in MC3T3‐E1 cells. NAC (ROS inhibitor) inhibited RIP1, RIP3, and increased ATF4; however, necrostatin‐1 (Nec‐1) (RIP1 inhibitor) specifically inhibited the protein expression of RIP1 and RIP3 and had no influence on ATF4. The use of antagomir‐214 suppressed the expression of miR‐214, RIP1, RIP3, and p‐MLKL, but increased ATF4 protein level in glucose and LPS‐induced cells. ATF4 knockdown by ATF4 small interfering RNA offset the effect of antagomir‐214. RIP1‐ and RIP3‐dependent necroptosis was confirmed in the inflamed gingival tissues of diabetes‐associated periodontitis patients and high glucose‐ and LPS‐ cotreated cells. It was suggested that miR‐214‐targeted ATF4 participated in the regulation of necroptosis in vivo and in vitro.
“…In addition, cellular death has also been reported in diabetic retinopathy, in age-related macular degeneration, and in programmed necrosis of the inflammatory cells, with all of them resulting from the action of AGEs, ROS, and MGO (Fig. 1 ) 6 , 9 . Fig.…”
Section: Introductionmentioning
confidence: 99%
“…Hyperglycemia is the trigger for the activation of several signaling pathways, and it represents a condition in which the cells become susceptible to necroptosis, apoptosis, and/or necrosis 6 . Both Type 1 and 2 diabetes mellitus (T1D and T2D) are metabolic disorders, apparently with distinct mechanisms, but with a significant loss of mass insulin-producing β-cells, due to cellular death 7 .…”
Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Several signaling pathways can be altered by having hyperglycemia in different tissues, producing oxidative stress, the formation of advanced glycation end products (AGEs), as well as the secretion of the pro-inflammatory cytokines and cellular death (pathological autophagy and/or apoptosis). However, the signaling pathways that are directly triggered by hyperglycemia appear to have a pivotal role in diabetic complications due to the production of reactive oxygen species (ROS), oxidative stress, and cellular death. The present review will discuss the role of cellular death in diabetic complications, and it will suggest the cause and the consequences between the hyperglycemia-induced signaling pathways and cell death. The signaling pathways discussed in this review are to be described step-by-step, together with their respective inhibitors. They involve diacylglycerol, the activation of protein kinase C (PKC) and NADPH-oxidase system, and the consequent production of ROS. This was initially entitled the “dangerous metabolic route in diabetes”. The historical usages and the recent advancement of new drugs in controlling possible therapeutical targets have been highlighted, in order to evaluate the evolution of knowledge in this sensitive area. It has recently been shown that the metabolic responses to stimuli (i.e., hyperglycemia) involve an integrated network of signaling pathways, in order to define the exact responses. Certain new drugs have been experimentally tested—or suggested and proposed—for their ability to modulate the possible biochemical therapeutical targets for the downregulation of retinopathy, nephropathy, neuropathy, heart disease, angiogenesis, oxidative stress, and cellular death. The aim of this study was to critically and didactically evaluate the exact steps of these signaling pathways and hence mark the indicated sites for the actions of such drugs and their possible consequences. This review will emphasize, besides others, the therapeutical targets for controlling the signaling pathways, when aimed at the downregulation of ROS generation, oxidative stress, and, consequently, cellular death—with all of these conditions being a problem in diabetes.
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